Hypothesis

Pulsatile mTOR activity is necessary to retain the civilizational‑versus‑survival dial’s responsiveness in aging immune cells. Constant mTOR suppression (e.g., chronic rapamycin) locks the dial in a survival state, abolishing the dynamic range needed for effective pathogen response and tissue repair. We hypothesize that brief, timed mTORC1 activation pulses re‑engage the nutrient‑sensing machinery (Rag GTPases, lysosomal v‑ATPase, TSC2) and restore metabolic flexibility, thereby preserving naïve T‑cell pools and limiting inflammaging.

Mechanistic Rationale

1. Nutrient‑sensing oscillators – In young cells, Rag GTPases cycle between active and inactive states on the lysosomal surface in response to amino‑acid fluxes, generating mTORC1 activity bursts that drive anabolic programs followed by autophagy‑mediated cleanup. Aging disrupts this cycle via constitutive Rag‑Ragulator signaling and impaired TSC2 GAP activity, producing starvation‑insensitive mTORC1 (1).
2. Pulse‑driven reset – A short leucine or glucose bolus during a fasting window transiently activates Rag‑mediated mTORC1 signaling, which in turn phosphorylates ULK1 to fine‑tune autophagy flux and stimulates S6K‑dependent feedback that restores TSC2 sensitivity. Repeating this pulse every 24‑48 h re‑trains the oscillator without causing chronic growth signaling.
3. Immune outcome – Pulsatile mTORC1 maintains a metabolic switch that lets effector T cells glycolyze during infection while prompting memory precursors to engage fatty‑acid oxidation and autophagy, preserving the naïve pool and reducing PD‑1/KLRG1 expression. Simultaneously, macrophage polarization oscillates between M1‑like antimicrobial and M2‑like reparative states, limiting SASP accumulation.

Testable Predictions

• Prediction 1: Mice receiving an intermittent leucine pulse (0.5 g/kg) at the start of each 16‑h fast will show higher naïve CD62L^+ CD44^− T‑cell frequencies and lower serum IL‑6 after 12 months compared with (a) ad libitum fed controls, (b) chronic rapamycin‑treated mice, and (c) continuous fasting mice.
• Prediction 2: Ex vivo stimulation of splenocytes from pulsed mice will produce a greater fold‑increase in IFN‑γ upon anti‑CD3/CD28 crosslinking and a more rapid return to baseline phosphorylation of S6K after stimulus removal, indicating restored mTORC1 dynamism.
• Prediction 3: Pharmacological blockade of Ragulator (using RagC‑dominant negative) will abolish the immune benefits of the leucine pulse, confirming that the effect depends on lysosomal mTORC1 sensing rather than downstream metabolic changes alone.

Experimental Outline

• Cohorts: young (3 mo) and aged (20 mo) C57BL/6 mice, n=15 per group.
• Interventions: (1) ad libitum, (b) chronic rapamycin (4 ppm diet), (c) 16:8 intermittent fasting (IF), (d) IF + leucine pulse (0.5 g/kg in drinking water at fast onset), (e) continuous IF without pulse.
• Readouts at 3, 6, 12 mo: flow cytometry for naïve/memory T‑cell subsets, exhaustion markers, macrophage CD86/CD206 ratios, serum cytokines (IL‑6, TNF‑α), and frailty index.
• Mechanistic assays: lysosomal Rag‑GTP loading (immunoprecipitation), phospho‑S6K and phospho‑ULK1 kinetics after ex vivo amino‑acid challenge, Seahorse metabolic flux (glycolysis vs OXPHOS).

If pulsed mTORC1 rescues immune function while constant suppression does not, the hypothesis supports the view that longevity depends on preserving a responsive civilization‑versus‑survival dial, not merely turning it down.